Project Summary Poly-ADP-ribosylation (PARylation) is a protein posttranslational modification (PTM) that was first documented in 1963. PARylation is catalyzed by a family of enzymes called Poly-ADP-ribose polymerases (PARPs). In particular, PARP1 is a nuclear protein that is activated as a result of sensing DNA strand breaks. The PARylation level in a quiescent cell is usually very low. In response to genotoxic stress, PARP1 is recruited to nicked DNA and is rapidly activated, resulting in the synthesis of a large number of PARylated proteins and initiation of the DNA damage repair mechanisms. The critical roles of PARP1 in mediating DNA repair and also cell death provide the rationale for developing PARP1 inhibitors to treat a number of human diseases, including cancer and ischemic stroke. In particular, cancer cells with defects in double-strand break (DSB) repair, such as BRCA1/2-mutated cells, are reliant on PARP1 activity for genome integrity. These cells undergo unsustainable genetic damage upon PARP1 inhibition. Indeed, late-stage clinical studies revealed that PARP1 inhibitor treatment significantly prolonged progression-free survival of BRCA-deficient ovarian cancer patients. This led to its recent approval by the FDA. Contrary to the fruitful efforts of characterizing the upstream regulators of PARP1, its genuine downstream targets are poorly defined, which has significantly hampered its further functional study. In particular, site-localization of protein PARylation remains a daunting challenge, due to its labile and heterogeneous nature. To address these pressing questions, we developed a large-scale mass spectrometric approach towards comprehensive characterization of the Asp- and Glu-PARylated proteome. We identified a total of 1,048 unique, endogenously modified D/E-PARylation sites on 340 proteins. These proteins are involved in not only DNA damage repair, but also a surprisingly wide array of other nuclear functions. Using a quantitative mass spectrometry experiment, we also identified many previously unknown PARP1 downstream targets, whose PARylation is sensitive to clinically relevant PARP1 inhibitors. In this proposal, we will leverage these preliminary results to continue to characterize protein ADP-ribosylation, with a long term goal of comprehensively understanding the role of PARPs and ADP-ribosylation in various pathophysiological processes. The specific aims of our proposed work are to: (1) develop a large-scale approach to site-specific characterization of the D/E-mono-ADP-ribosylated proteome; (2) develop a large- scale method to measure absolute protein PARylation stoichiometries; and (3) develop a chemoproteomic approach to systemically investigating the specificity of clinically relevant PARP1 inhibitors. We will accomplish our goals with a multi-disciplinary approach, utilizing tools including proteomics, chemical biology, biochemistry, bioinformatics and molecular biology. Knowledge we garner in this proposal will also have a profound impact on how to further explore PARPs as potential therapeutic targets for treating human diseases.